The objective of this study is to characterize quantitatively the basic mechanisms underlying pacemaker activity in mammalian neurons (e.g., motoneurons) an heart cells, and to determine how this activity is regulated. Neurons obtained from rat embryos and heart cells obtained from rat pups will be grown in dissociated cell culture to provide preparations with direct access to the membrane of individual cells. The voltage and time-dependence of the pacemaker currents will be mapped using voltage clamp techniques, and the ion selectivity of individual current systems will be determined using a rapid microperfusion system to change the ion concentrations bathing the cell membrane. A new isolated membrane preparation, consisting of an isolated patch of cell membrane 'glued' to the orifice of a glass capillary tube, will be used to study the role of intracellular variables such as calcium and cyclic nucleotide concentrations in regulating the membrane pacemaker currents. This preparation will allow rapid, precise control of the solutions bathing the inner side of the membrane, as well as good voltage clamp control and membrane current measurements. Preliminary data indicate that the development of the membrane current systems as well as the axonal growth of spinal motoneurons require at least one high molecular weight growth of differentiation factor that is present in medium conditioned by exposure to skeletal muscle cells. We propose to isolate growth factor(s) from this conditioned medium and to characterize its effects on the development of motoneurons and other cell types. We will determine whether this factor(s) is related to the classical nerve growth factor required by sympathetic ganglion neurons, or to a recently discovered choline acetyltransferase enchancing factor.